ABSTRACT As cells exit mitosis, they pause at the abscission checkpoint to ensure that the mitotic program has been completed successfully. They then proceed through abscission, irreversibly separating the two daughter cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery plays essential roles in both of these important cytokinetic processes. Certain ESCRT components are negatively regulated by the abscission checkpoint to prevent premature abscission. Once the checkpoint is satisfied, the ESCRT machinery then assembles in the midbody to constrict the membrane and carry out abscission. Projects in this proposal are designed to uncover the molecular mechanisms that underlie key steps in these processes. Specifically, we will: characterize the regulation of ALIX, a key ESCRT factor that nucleates assembly of constricting ESCRT-III filaments within the midbody (AIM 1), determine how ESCRT-III filaments recruit MIT domain- containing proteins to the midbody and define how these proteins function in cytokinesis (AIM 2), and characterize the structures and functions of the 9 related ?meiotic clade? AAA ATPases that work together to promote abscission by remodeling midbody microtubules and ESCRT-III filaments. Structural studies in Subaim 1.1 will target the different conformational states along the ALIX activation pathway, with the goal of learning how mitotic phosphorylation activates ALIX to participate in abscission. These studies will build on our previous biochemical and structural analyses of the ALIX core domains, both free and in complex with ESCRT-III ligands. Cell biological studies in Subaim 1.2 will define how checkpoint activation delays ALIX recruitment to the midbody and test whether ALIX sequestration inhibits abscission. Preliminary studies have established the delay in ALIX recruitment and shown that checkpoint activation causes ALIX to concentrate within cytoplasmic foci, together with other factors required for abscission and the abscission checkpoint. In Subaim 2.1, we will identify and structurally characterize complexes of the 25 different human MIT domain proteins with their binding sites on the tails of the 12 different ESCRT-III proteins. To date, these studies have revealed more than 20 new interactions and produced six structures of ESCRT-III-MIT complexes. Complementary studies in Subaim 2.2 will identify human MIT proteins required for different stages of cytokinesis and characterize their functions. These approaches have already identified three new MIT proteins with important roles in the abscission checkpoint. Structural studies in Subaim 3.1 will target microtubule severing AAA ATPases in complex with relevant substrates. These studies will complement our recent high resolution cryoEM structure of the related Vps4 AAA ATPase in complex with an ESCRT-III substrate. Finally, biochemical studies in Subaim 3.2 will examine how these related enzymes discriminate between ESCRT-III filaments and microtubule substrates, and test our hypothesis that these ATPases utilize a common mechanism to translocate and unfold polypeptide substrates. These studies will build upon our reconstitution and high resolution cryoEM structure of helical double stranded filaments formed by the ESCRT-III proteins CHMP1B and IST1, both free and in complex with membranes. Taken together, our studies will define the molecular mechanisms that underlie a series of central events in the fundamental cell biology of cytokinesis.